Establishing the pathogenicity of novel mitochondrial DNA sequence variations: a cell and molecular biology approach

Tese de doutoramento em Ciências da Saúde, no ramo de Ciências Biomédicas, apresentada à Faculdade de Medicina da Universidade de CoimbraAs doenças mitocondriais são das doenças metabólicas mais frequentes e representam um grande encargo económico na sociedade. Atualmente, existem mais de 60 mutações no DNA mitocondrial (mtDNA) associadas a patologias. Uma vez que o mtDNA é altamente polimórfico e apresenta características peculiares, a patogenicidade de uma nova alteração detetada, apoia-se numa série de critérios para estabelecer a correlação genótipo-fenótipo. O presente trabalho inclui o estudo de quatro variações de sequência no mtDNA, não classificadas, com potencial efeito deletério, identificadas em quatro doentes portugueses com suspeita de doença mitocondrial e sem parentesco, estudados no Laboratório de Bioquímica Genética, Centro de Neurociências e Biologia Celular e Molecular – Universidade de Coimbra. O doente 1 (P1) apresentou neuropatia ótica bilateral grave. O doente 2 (P2) manifestou deficiência mental grave, atrofia cerebelar, ataxia grave, fácies grosseira, macrocefalia relativa, hipotonia congénita, afasia, e outras características tal como clinodactilia. O doente 3 (P3) apresentou oftalmoplegia externa progressiva crónica. O doente 4 (P4) sofreu morte súbita infantil, com a suspeita posterior de cardiomiopatia. Foi identificada uma variante no mtDNA em cada doente em genes que codificam subunidades dos complexos enzimáticos da fosforilação oxidativa ou RNAs de transferência. As variantes identificadas foram m.8418T>C, p.Leu18Pro (MT-ATP8), m.14771C>A, p.Pro9Thr (MT-CYB), m.7486G>A, mt-tRNASer(UCN) (MT-TS1), m.14706A>G, mt-tRNAGlu (MT-TE), nos doentes 1, 2, 3 e 4, respetivamente. Foram realizados estudos biomoleculares, usando uma abordagem de genómica funcional para avaliar a patogenicidade das variantes no mtDNA de forma a estabelecer o diagnóstico genético dos quatro doentes e clarificar o mecanismo de patogenicidade. Os métodos usados incluíram sequenciação de Sanger, pirosequenciação, sequenciação de nova geração, PCR longo e PCR em tempo real, histologia, histoquímica, cultura de células, espectrofotometria, fluorimetria, Western-blot, eletroforese nativa em gel de acrilamida, tecnologia Seahorse Bioscience® e microscopia de transmissão eletrónica. As amostras investigadas foram obtidas a partir da cultura primária de fibroblastos (derivada de biópsia de pele) dos quatro doentes descritos acima e de três controlos, além de sangue (estudo familiar dos P2 e P4, e controlos), músculo (P3 e P4) e fígado (P4). A avaliação funcional nas células do P1 mostrou um decréscimo nos níveis da proteína A6L (codificada pelo gene MT-ATP8) do complexo V, uma redução do assembly do complexo V, disfunção mitocondrial (nomeadamente alterações na atividade enzimática da cadeia respiratória mitocondrial, consumo de oxigénio, glicólise, níveis de ATP intracelulares, potencial de membrana mitocondrial e produção de espécies reativas de oxigénio), com evidencias de stresse no reticulo endoplasmático. Nas células do P2 foi detetada disfunção mitocondrial, com diminuição dos níveis da proteína citocromo b e da atividade do complexo III. Observou-se também a presença de corpos multilamelares, sugerindo um comprometimento da autofagia. Os fibroblastos do P3 apresentaram redução no assembly dos quatro complexos com subunidades codificadas pelo mtDNA, disfunção mitocondrial, alterações no potencial de membrana mitocondrial e na produção de espécies reativas de oxigénio, bem como a presença de corpos multilamelares. Foram ainda detetadas evidências histoquímicas e morfológicas, sugestivas de doença mitocondrial, no músculo. No P4, a deficiência na atividade enzimática da fosforilação oxidativa foi detetada apenas na biópsia de fígado e músculo, o que sugere a existência de especificidade tecidular. A redução no assembly dos quatro complexos com subunidades codificadas pelo mtDNA foi o resultado mais significativo, além do aumento anormal do tamanho das mitocôndrias. Para além disso, a investigação genética revelou uma segunda alteração (conhecida como “deleção comum”) no mtDNA (P3), descrita em associação com a patologia. Foi ainda detetada uma mutação no gene SNX14 e outra no gene MYBPC3 nos P3 e P4, respetivamente, em estudos paralelos realizados por colaboradores. O trabalho permitiu: (i) confirmar o potencial patogénico das quatro variantes no mtDNA; (ii) reportar variantes com evidências funcionais de patogenicidade; (iii) incluir estas variantes na investigação genética de doentes que apresentem fenótipo semelhante; (iv) contribuir para desenvolvimentos significativos na patogenicidade em doenças mitocondriais. Em conclusão, as evidências sugerem que: (i) as variantes analisadas estão provavelmente envolvidas na disfunção mitocondrial; (ii) défice mitocondrial pode influenciar mecanismos celulares, nomeadamente stresse no reticulo endoplasmático e a macroautofagia; (iii) as doenças mitocondriais são heterogéneas e complexas, com possível dupla origem genética, além das associadas a síndromes de depleção/deleção, já conhecidas. Abstract Mitochondrial dis orders are among the most frequent metabolic disorders and a major burden for society. There are more than 60 con firmed mitochondrial DNA (mtDNA) point mutations associated with several diseases. Since t he mtDNA is highly polymorphic with pe culiar properties, the pathogenicity of a novel sequence variation needs to be determined using a series of criteria , including functional studies, for establish ing genotype/phenotype correlation s . The present study comprises four unclassified mtDNA variants with potential pathogenic effect , identified in four unrelated P ortuguese patients suspected of mitochondrial disease, at Lab oratory of Biochemical Genetics, C enter for neuroscience and cell biology – University of Coimbra . Patient 1 (P1) presented se vere bilateral optic neuropathy. P atient 2 (P2) manifested severe intellectual disability, cerebella r a trophy, severe ataxia, coa r se face, relative macrocephaly, congenital hypotonia, absent speech, and other features such as clinodactyly. Patient 3 (P3) presented chronic progressive external ophthalmoplegia. Finally, patient 4 (P4) was suspected of cardiomyopathy after sudden death. A mtDNA variant has been identified in each patient, affecting genes encoding subunits of oxidative phosphorylation (OXPHOS) enzymatic complexes or variants in mt - tRNA genes. The alterations identified were m.8418T>C, p.Leu18Pro ( MT - ATP8 ), m.14771C>A, p.P ro9Thr ( MT - CYB ), m.7486G>A, mt - tRNA Ser(UCN) ( MT - TS1 ) m.14706A>G, mt - tRNA Glu ( MT - TE ), in patients 1, 2, 3 and 4, respectively. Accordingly, a series of biomolecular studies, using a functional genomics’ approach was conducted for evalu ating the pathogenic ity of the unclassified mtDNA variants, for establish ing the genetic diagnosis of the patients and clarify the pathogenic mechanism. The methods used included Sanger s equencing, pyrosequencing, next generation sequencing, long - range PCR, real - time PCR, his tochemistry, histology, cell culture, western - blot, blue native polyacrylamide gel electrophoresis , spectrophotometry, fluorimetry, Seahorse Bioscience ® technology and tra nsmission electron microscopy. The samples investigated included cultured fibroblasts (derived from skin biopsy) of the four patients described above plus three controls, and blood (family studies of P2 and P4 , and controls ) , muscle (P3 and P4) and liver (P4) samples when available. The functional evaluation in the skin fibroblasts of P1 showed a decrease in A6L protein level of complex V (encoded by MT -ATP8 gene), a reduction of the fully assembled complex V, mitochondrial dysfunction (namely alterations in OXPHOS enzymatic activity and oxygen consumption, glycolysis, intracellular ATP levels, mitochondrial membrane potential and reactive oxygen species production) and evidences of endoplasmic reticulum stress. In the cells of P2, a decrease in the cytochrome b levels and activity of complex III was observed. Moreover, in addition to mitochondrial dysfunction detected, the presence of multilamellar bodies were identified in skin fibroblasts, suggesting autophagy impairment. Skin fibroblasts of P3 presented a reduction in the assembly of the four complexes with subunits encoded by mtDNA, mitochondrial dysfunction, changes in mitochondrial membrane potential and in the production of reactive oxygen species. Also, multilamellar bodies were observed in fibroblasts. Additionally, morphological and histochemical evidences for mitochondrial disease were detected in patient’s muscle. In P4, deficiency of OXPHOS enzymatic activity was only observed in muscle and liver biopsy, suggesting tissue specificity. The reduction in assembly of the four complexes with subunits encoded by mtDNA was the more significant finding detected in patient’s fibroblasts, in addition to abnormal increase of mitochondria’s size. Furthermore, deep er molecular genetic investigation revealed a second mtDNA alteration in P 3, described in association with the pathology, known as “ common deletion”. Also, a muta tion in SNX14 gene (P2) and a mutation in MYBPC3 gene (P4) were detected, in parallel studies performed by other collaborators. The present work allowed: (i) to confirm the high pathogenic potential of the four unclassified mtDNA variants; (ii) to report the variants showing functional evidences for its pathogenicity; (iii) to include these sequence variations in the genetic investigation of the patients presenting similar phenotypes; (iv) to contribute for significant developments in the field of mitochondrial diseases pathogenicity. In conclusion, the evidences taken together suggest that: (i) the mtDNA variants analysed are probably involved in the observed mitochondrial dysfunction; (ii) mitochondrial impairment may influence cellular mechanisms, namely endoplasmic xli reticulum stress and macroautophagy; (iii) mitochondrial diseases are heterogeneous and complex diseases that may have a double genetic origin, besides the known depletion or deletion - associated syndromes.Mitochondrial disorders are among the most frequent metabolic disorders and a major burden for society. There are more than 60 confirmed mitochondrial DNA (mtDNA) point mutations associated with several diseases. Since the mtDNA is highly polymorphic with peculiar properties, the pathogenicity of a novel sequence variation needs to be determined using a series of criteria, including functional studies, for establishing genotype/phenotype correlations. The present study comprises four unclassified mtDNA variants with potential pathogenic effect, identified in four unrelated Portuguese patients suspected of mitochondrial disease, at Laboratory of Biochemical Genetics, Center for neuroscience and cell biology – University of Coimbra. Patient 1 (P1) presented severe bilateral optic neuropathy. Patient 2 (P2) manifested severe intellectual disability, cerebellar atrophy, severe ataxia, coarse face, relative macrocephaly, congenital hypotonia, absent speech, and other features such as clinodactyly. Patient 3 (P3) presented chronic progressive external ophthalmoplegia. Finally, patient 4 (P4) was suspected of cardiomyopathy after sudden death. A mtDNA variant has been identified in each patient, affecting genes encoding subunits of oxidative phosphorylation (OXPHOS) enzymatic complexes or variants in mt-tRNA genes. The alterations identified were m.8418T>C, p.Leu18Pro (MT-ATP8), m.14771C>A, p.Pro9Thr (MT-CYB), m.7486G>A, mt-tRNASer(UCN) (MT-TS1) m.14706A>G, mt - tRNAGlu (MT-TE), in patients 1, 2, 3 and 4, respectively. Accordingly, a series of biomolecular studies, using a functional genomics’ approach was conducted for evaluating the pathogenicity of the unclassified mtDNA variants, for establishing the genetic diagnosis of the patients and clarify the pathogenic mechanism. The methods used included Sanger sequencing, pyrosequencing, next generation sequencing, long-range PCR, real-time PCR, histochemistry, histology, cell culture, western-blot, blue native polyacrylamide gel electrophoresis, spectrophotometry, fluorimetry, Seahorse Bioscience® technology and transmission electron microscopy. The samples investigated included cultured fibroblasts (derived from skin biopsy) of the four patients described above plus three controls, and blood (family studies of P2 and P4, and controls), muscle (P3 and P4) and liver (P4) samples when available. The functional evaluation in the skin fibroblasts of P1 showed a decrease in A6L protein level of complex V (encoded by MT-ATP8 gene), a reduction of the fully assembled complex V, mitochondrial dysfunction (namely alterations in OXPHOS enzymatic activity and oxygen consumption, glycolysis, intracellular ATP levels, mitochondrial membrane potential and reactive oxygen species production) and evidences of endoplasmic reticulum stress. In the cells of P2, a decrease in the cytochrome b levels and activity of complex III was observed. Moreover, in addition to mitochondrial dysfunction detected, the presence of multilamellar bodies were identified in skin fibroblasts, suggesting autophagy impairment. Skin fibroblasts of P3 presented a reduction in the assembly of the four complexes with subunits encoded by mtDNA, mitochondrial dysfunction, changes in mitochondrial membrane potential and in the production of reactive oxygen species. Also, multilamellar bodies were observed in fibroblasts. Additionally, morphological and histochemical evidences for mitochondrial disease were detected in patient’s muscle. In P4, deficiency of OXPHOS enzymatic activity was only observed in muscle and liver biopsy, suggesting tissue specificity. The reduction in assembly of the four complexes with subunits encoded by mtDNA was the more significant finding detected in patient’s fibroblasts, in addition to abnormal increase of mitochondria’s size. Furthermore, deeper molecular genetic investigation revealed a second mtDNA alteration in P3, described in association with the pathology, known as “common deletion”. Also, a mutation in SNX14 gene (P2) and a mutation in MYBPC3 gene (P4) were detected, in parallel studies performed by other collaborators. The present work allowed: (i) to confirm the high pathogenic potential of the four unclassified mtDNA variants; (ii) to report the variants showing functional evidences for its pathogenicity; (iii) to include these sequence variations in the genetic investigation of the patients presenting similar phenotypes; (iv) to contribute for significant developments in the field of mitochondrial diseases pathogenicity. In conclusion, the evidences taken together suggest that: (i) the mtDNA variants analysed are probably involved in the observed mitochondrial dysfunction; (ii) mitochondrial impairment may influence cellular mechanisms, namely endoplasmic reticulum stress and macroautophagy; (iii) mitochondrial diseases are heterogeneous and complex diseases that may have a double genetic origin, besides the known depletion or deletion-associated syndromes.FEDER - COMPETE2020 (Strategic projects: POCI-01-0145-FEDER-007440, HealthyAging2020 CENTRO-01-0145-FEDER000012-N2323 and New Strategies to Manage Brain Diseases 2013 to 2015| CENTRO07-ST24-FEDER-002002/6/8 Programa Operacional Regional do Centro - Projecto Mais Centro